US5124264A - Monoclonal antibody recognizing atrial natriuretic polypeptide - Google Patents

Monoclonal antibody recognizing atrial natriuretic polypeptide Download PDF

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US5124264A
US5124264A US07/314,930 US31493089A US5124264A US 5124264 A US5124264 A US 5124264A US 31493089 A US31493089 A US 31493089A US 5124264 A US5124264 A US 5124264A
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anp
antibody
hanp
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ranp
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Hiroo Imura
Kazuwa Nakao
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Shionogi and Co Ltd
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    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

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  • the present invention relates to a monoclonal antibody which recognizes the N-terminal of atrial natriuretic polypeptide (hereinafter referred to as ANP), a hybridoma which produces the monoclonal antibody, and a method for immunoassay of ANP with the use of the monoclonal antibody.
  • ANP atrial natriuretic polypeptide
  • ANP is a polypeptide contained in granules produced by atrial myocyte, and exerts a natriuretic action as well as a strong diuretic action.
  • Atrial natriuretic polypeptide A series of polypeptides called atrial natriuretic polypeptide (ANP) as a whole have been isolated from human and rat atrial tissues, and it has been suggested that the polypeptides are associated with the homeostasis of body fluid and the control of blood pressure (Kangawa, K. et al, Biochem. Biophys. Res. Commun., 118, 131-139, 1984).
  • Such polypeptides are found not only in humans but also in rats, and called hANP and rANP respectively.
  • the hANP and rANP are each subclassified into three types, namely, ⁇ , ⁇ , and ⁇ .
  • human ANP of ⁇ -type and rat ANP of ⁇ -type are abbreviated as ⁇ -hANP and ⁇ -rANP respectively.
  • ANP When it is not necessary to specify the source or subclass of ANP, it is simply referred to as "ANP".
  • ⁇ -hANP consists of 28 amino acid residues. Cys[7], i.e., Cys at the 7th position from the N-terminal, forms a disulfide linkage with Cys[23], i.e., Cys at the 23rd position, and therefore the peptide sequence between Cys[7] and Cys[23] forms a ring structure (Biochem. Biophys. Res. Commun., 118, 131-139, 1984). ⁇ -hANP is different from ⁇ -rANP in that the amino acid residue at the 12th position from the N-terminal is Met in the former, while it is Ile in the latter (Biochem. Biophys. Res. Commun., 117, 839-865, 1983).
  • ⁇ -hANP is an antiparallel dimer of ⁇ -hANP (Japanese Patent Unexamined Publication No. 184098/1985).
  • ⁇ -hANP consists of 126 amino acid residues, and its 99-126 amino acid sequence on the C-terminal exactly corresponds to ⁇ -hANP.
  • ANP is also present in the central nervous system (Morii, N. et al, Biochem. Biophys. Res., Commun., 127, 413-419, 1985), and that the major molecules of ANP in the brain and spinal cord of rats are ⁇ -rANP [4-28] and ⁇ -rANP [5-28] (Shiono, S. et al, Biochem. Biophys. Res. Commun., 135, 728-734, 1986; Morii, N. et al, ibid. 145, 196-203, 1987).
  • ANP in the brain and spinal cord functions as a neuropeptide
  • ANP in the circulating blood functions as a hormone
  • the afore-mentioned polyclonal rabbit antiserum against ⁇ -ANP can not distinguish ANP in the circulating blood from ANP in the brain and spinal cord, but it has been used in various ways.
  • the antiserum was used in immunohistochemical studies (Kawata, M. et al, Neuroscience 16, 521-546, 1985).
  • the antiserum was injected into the cerebral ventricle of rat to neutralize the action of endogenous ANP in the brain, thereby water intake was enhanced (Katsuura, G. et al, European J. Pharmacol. 121, 285-287, 1986).
  • polyclonal antibody to ANP contained in the antiserum are useful in various aspects.
  • the antiserum has unavoidable disadvantages in that it has short supply, it contains various antibodies recognizing a variety of epitopes and it contains unnecessary antibodies against antigens other than ANP.
  • a monoclonal antibody named llA-All which recognizes ⁇ -hANP is known.
  • the monoclonal antibody was obtained using atriopeptin II, a kind of rat ANP, as an immunogen, and its epitope is positioned between Cys[7] and Ser[25] constituting disulfide linkage. That is to say, it is considered that the epitope exists in the ring structure of ANP.
  • the monoclonal antibody has the same affinity to hANP (Met 12) and rANP (Ile 12), and it recognizes both rANP and hANP (Life Science 38, 1991-1997, 1986).
  • the present invention provides a novel monoclonal antibody recognizing N-terminal sequence of ⁇ -ANP. More particularly, it provides a monoclonal antibody which specifically recognizes ANP in circulating blood that consists of 28 amino acid residues.
  • the monoclonal antibody of the invention recognizes mainly 2-3 amino acid residues on the N-terminal of ⁇ -ANP, and furthermore, there is a possibility that a part of the ring structure of ⁇ -ANP is included in its epitope.
  • the monoclonal antibody of the invention recognizes both ⁇ -hANP and ⁇ -rANP because the difference between ⁇ -hANP and ⁇ -rANP lies only in the 12th amino acid residue from the N-terminal.
  • the antibody of the invention permits the measurement of ⁇ -ANP which exists only in the circulating blood because ⁇ -ANP of the central nervous system- lacks N-terminal.
  • the monoclonal antibody of the present invention has the highest affinity with ligand, and has a binding specificity different from other known antibodies. Moreover, the monoclonal antibody of the invention can also be used for immunohistochemical and neutralizing tests in rats. Besides, the antibody of the invention allows highly sensitive measurement of ⁇ -ANP in RIA. Furthermore, when the antibody of the invention is used in combination with an antibody recognizing the C-terminal of ⁇ -ANP, such as polyclonal antiserum against ⁇ -ANP[17-28], it is possible to conduct sandwich enzyme immunoassay (EIA) with very high sensitivity, and in particular, it is possible to measure only ⁇ -ANP circulating in the blood. In conventional methods of measuring ⁇ -ANP, it has been necessary to purify ⁇ -ANP from a sample of plasma, etc. However, in the method of the present invention, such purification is not necessary owing to its high sensitivity.
  • EIA sandwich enzyme immunoassay
  • the monoclonal antibody of the invention which recognizes the N-terminal sequence of ⁇ -ANP, is very useful in the study of physiological or pathophysiological significance of ANP as a hormone.
  • ⁇ -hANP is a polypeptide consisting of 28 amino acid residues, and its molecular weight is too low to sufficiently induce antibody-production (low- immunogenicity). For this reason, ⁇ -hANP is conjugated with any other protein of higher molecular weight, such as bovine serum albumin or bovine thyroglobulin, when it is used as an immunogen.
  • the conjugate thus obtained is emulsified in a suitable adjuvant, such as Freund's complete adjuvant, and then is used to immunize mice.
  • Immunization is performed by repeatedly inoculating the above emulsion into mice at intervals of several weeks intraperitoneally, subcutaneously or intravenously. Three to five days after the last immunization, the spleen is taken out, which is used as a source providing antibody-producing cells.
  • myeloma cells having a suitable marker such as hypoxanthine-guanine-phosphoribosyl transferase-deficiency (HGPRT - ) or thymidine kinase deficiency (TK - ) are prepared.
  • HGPRT - hypoxanthine-guanine-phosphoribosyl transferase-deficiency
  • TK - thymidine kinase deficiency
  • a culture medium for the hybridoma growth there may be employed such media as Eagle's MEM, Dulbecco's modified medium, or RPMI-1640, with the addition of about 15% fetal calf serum (FCS), although the medium is not limited thereto.
  • FCS fetal calf serum
  • the myeloma cells and the spleen cells are mixed at the ratio of about 1:6 in the presence of a fusing agent.
  • a fusing agent 50% polyethylene glycol (PEG) is generally employed because of its high fusing efficiency.
  • Fused cells are selected by HAT selection method.
  • the hybridomas contained in the culture supernatant are screened according to conventional methods such as membrane fluorescence antibody technique, enzyme linked immunosorbent assay (ELISA method), immunological tissue staining method, and RIA, to select aimed hybridomas capable of secreting desired immunoglobulin.
  • re-cloning is conducted as follows: normal spleen cells are placed as a feeder layer in a 96-well microplate and the selected hybridomas are placed thereon at a rate not exceeding one piece per well, and screening is performed again on cultured clones. Homogeneous hybridomas are obtained by repeating such sub-cloning process.
  • the hybridomas obtained above are cultured in vitro or in vivo to prepare a monoclonal antibody of the present invention.
  • conventional media such as mentioned above may be used with addition of FCS.
  • the monoclonal antibody is obtained from the culture supernatant.
  • the hybridoma is inoculated into the abdominal cavity of a mammal. One to two weeks after that, ascites fluid is collected, from which monoclonal antibody is obtained.
  • in vivo culture produces a far larger quantity of antibody, and therefore, is preferred.
  • the monoclonal antibody obtained from culture supernatant or ascites fluid is purified by known methods, such as ammonium sulfate fractionation, adsorption to Protein A column and DEAE Sepharose column chromatography, or a combination thereof.
  • the inventors have obtained a monoclonal antibody to ⁇ -ANP in accordance with the process as mentioned above, which was designated as KY-ANP-II-, and have examined its characteristics.
  • a weak cross reactivity with ⁇ -rANP[3-28] was also observed.
  • the antibody did not react with ⁇ -rANP[4-28] nor with ⁇ -rANP[5-28]. This indicates that 2-3 amino acid residues of the N-terminal of ⁇ -ANP is most important for the recognition by the antibody.
  • ⁇ -hANP which has a structure corresponding to ⁇ -hANP plus additional amino acids attached to the N-terminal of the ⁇ -hANP.
  • this antibody recognizes ⁇ -rANP as well as ⁇ -hANP, which shows that 12th amino acid on ⁇ -ANP does not constitute the epitope.
  • 2- 3 amino acids of the N-terminal have been found to be important, ⁇ -ANP[1-11] was not sufficiently recognized by this antibody.
  • ANPs in the central nervous system of rats mostly lack several amino acids at the N-terminal of ⁇ -ANP, as illustrated by ⁇ -ANP[4-28] and ⁇ -ANP[5-28]. As such, they are different from the ⁇ -ANP present in the circulatory system. Therefore, the monoclonal antibody of the present invention is highly specific for ANP in the circulating blood, and it equally recognizes both human and rat ⁇ -ANPs.
  • the hybridoma KY-ANP-II which produces the monoclonal antibody, KY-ANP-II, of the present invention has been deposited with the Fermentation Research Institute, Agency of the Industrial Science & Technology, Higashi 1-1-3, Tsukuba City, Ibaraki Prefecture, Japan, since Feb. 2, 1988, under the name of Mouse hybridoma KY-ANP-II, Bikoken Joki No. 1695 (FERM BP-1695), in compliance with the Budapest Treaty.
  • RIA which employs a single antibody, or sandwich EIA may be mentioned.
  • a competitive method may be mentioned in which, as shown in the Example hereinafter described, a sample to be tested or a standard ⁇ -ANP and a predetermined amount of isotope-labeled ⁇ -ANP are allowed to competitively bind to the antibody, and the radioactivity bound to the antibody is measured.
  • Sandwich EIA is conducted employing the antibody KY-ANP-II in combination with other antibody, for instance, the afore-mentioned antiserum CR-3, which recognizes the C-terminal of ⁇ -ANP, in such a manner as mentioned below.
  • a solid phase for immobilizing the antibody there may be used such carriers as glass or plastic beads or balls, tubes and plates, which are commercially available and are generally used in immunoassay as a carrier for an antigen-antibody reaction.
  • An antibody recognizing N-terminal or C-terminal of ⁇ -ANP is adsorbed to any of these carriers. The adsorption is usually performed by allowing the antibody to contact with the carrier overnight in a phosphate buffer at pH 6-10, preferably at around neutral pH, at room temperature.
  • the carrier on which the antibody has been adsorbed is stored in a cold place in the presence of an antiseptic agent such as sodium azide.
  • Both monoclonal antibody and polyclonal antibody can be used in the above procedure. Separation and purification of the antibody to be employed in the above procedure can be conducted in the following manner.
  • Ascites or antiserum containing the antibody is fractionated with sodium sulfate, and then passed through a DEAE-cellulose column, whereby IgG is obtained.
  • the IgG thus obtained is digested with pepsin to make F(ab') 2 fragment, which is then reduced with 2-mercaptoethylamine to obtain the desired anti- ⁇ -hANP Fab'.
  • Preparation of Fab' from IgG is detailed in J. Immunoassay, 4, 209-327 (1983), and the same procedure may be used in the present invention.
  • alkaline phosphatase As an enzyme to be employed for the purpose of labelling the antibody, there may be used alkaline phosphatase, ⁇ -D-galactosidase, peroxidase, glucose oxidase, etc. In the present invention, however, it is particularly preferable to use horseradish peroxidase.
  • a bridging agent which is used to conjugate the enzyme with the antibody
  • the reaction of the bridging agent with the enzyme and the antibody is performed in accordance with conventional methods with necessary modification depending on the nature of particular bridging agent.
  • the fragment of the antibody such as Fab', Fab, and F(ab') 2
  • enzyme-labeled antibody can be polyclonal or monoclonal antibody. The purification of the enzyme-labeled antibody obtained by the use of the above-mentioned bridging agent by affinity chromatography provides a more highly sensitive immunoassay system.
  • the purified enzyme-labeled antibody is stored in a cold and dark place with addition of a stabilizer such as thimerosal or glycerin, or after being lyophilized.
  • an antibody recognizing N-terminal of ⁇ -hANP is immobilized where an antibody recognizing C-terminal is enzyme-labeled
  • an antibody recognizing C-terminal is immobilized where an antibody recognizing N-terminal is enzyme-labeled. Since immobilization of an antibody usually requires a large quantity of antibody, the immobilization of a monoclonal antibody, for instance, KY-ANP-II of the present invention which can be obtained steadily in a large quantity, is preferred. However, a polyclonal antibody prepared from antiserum can also be used without any inconvenience.
  • An antibody to be enzyme-labeled can also be either a monoclonal antibody or a polyclonal antibody, provided that the antibody recognizes a site different from that recognized by the immobilized antibody.
  • KY-ANP-II of the present invention can be used as an immobilized antibody, while the above-mentioned antiserum CR-3 can be used as an enzyme-labeled antibody, and vice versa.
  • a monoclonal antibody recognizing the C-terminal of ⁇ -hANP and antisera recognizing the N-terminal may also be utilized.
  • FIG. 1 shows Scatchard plot of the binding between [ 125 I] ⁇ -hANP and monoclonal antibody KY-ANP-II. Ascites containing KY-ANP-II was incubated with [ 125 I] ⁇ -hANP (2.7-140 pM, 500 ⁇ l/tube) for 48 hr. at 4° C., and specific binding was measured after separation with dextran-coated charcoal.
  • FIG. 2 shows a typical standard curve of ⁇ -hANP in RIA wherein KY-ANP-II is employed, and cross reaction curves of related peptides.
  • [ 125 I] ⁇ -hANP and standard ⁇ -hANP or related peptides in various concentrations were incubated with KY-ANP-II for 24 hr. at 4° C.
  • the symbols employed have the following meanings: . . . ⁇ -hANP, ⁇ . . . ⁇ -rANP, . . . ⁇ -rANP(3-28), ⁇ . . . ⁇ -hANP(4-28) and ⁇ -rANP(4-28), . . .
  • Synthetic ⁇ -hANP (1.5 mg) and bovine thyroglobulin (5.4 mg) were dissolved in 2 ml of distilled water.
  • To this solution was added dropwise a solution of 30 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in 1 ml of distilled water over a period of 10 min. at room temperature, after which the mixture was stirred at room temperature for 24 hr. The mixture was then dialyzed 6 times against 3 l of distilled water over a period of 3 days. The dialyzate was divided into 5 portions, which were stored at -20° C. (See Biochem. Biophys. Res. Commun., 124, 815-821, 1984).
  • each of the stored solutions (each containing 300 ⁇ g of ⁇ -hANP) was added distilled water to make 1.2 ml, which was then suspended in 1.2 ml of Freund's complete adjuvant. About 2 ml portion was injected intraperitoneally and subcutaneously into 10 BALB/c female mice (200 ⁇ l per animal). The animals were boostered in the same manner 3 weeks later. After that, 10-30 ⁇ g of ⁇ -hANP emulsified in complete adjuvant was injected subcutaneously 3 times at intervals of 4 weeks. Four days after the last immunization with intravenous injection of 10 ⁇ g of ⁇ -hANP, the spleens of the mice were taken out, which were used for cell fusion.
  • Spleen cells (6 ⁇ 10 7 cells) and myeloma cells (X63-Ag8.653, 107 cells) were mixed in Dulbecco's medium (DMEM), and the mixture was centrifuged at 1500 rpm for 5 min. at 4° C. The pellets obtained were loosened by warming at 37° C., and then 1 ml of 50% PEG4000 (PEG 1 g/DMEM 1 ml) was added dropwise at 37° C. over a period of 1 min. The mixture was allowed to stand for 2 min. at 37° C., after which it was diluted by dropwise addition of 10 ml of DMEM at 37° C. over a period of 5 min. The mixture was then washed by centrifugation at 4° C. with DMEM containing 15% FCS.
  • DMEM Dulbecco's medium
  • the resultant hybridomas were selected in HAT medium containing 15% fetal calf serum.
  • the antibody production in the culture medium was examined periodically by RIA by the use of [ 125 I] ⁇ -hANP. Growth of hybridomas was observed in almost all wells, 8% (29 wells) of which produced antibodies.
  • Cells producing antibodies were cloned twice by the limiting dilution method with the use of mouse thymus cells as a feeder. A clone which produces an antibody having the strongest reactivity, i.e., KY-ANP-II, was established. In order to study the properties of KY-ANP-II, the clone was further cultivated.
  • mice were pretreated by intraperitoneally injecting 0.5 ml/animal of pristane twice at intervals of 1 to 2 weeks.
  • To each of the mice was intraperitoneally injected 5 ⁇ 10 6 cells of hybridoma KY-ANP-II suspended in 200 ⁇ l of DMEM.
  • Ascites taken from the mice was purified by means of Protein A-Sepharose CL-4B column to obtain monoclonal antibody KY-ANP-II.
  • the isotype of the monoclonal antibody obtained above was determined by Ouchterlony's method (Mouse Monoclonal Typing Kit, Miles).
  • the affinity constant was determined by Scatchard method by means of the RIA to be mentioned later.
  • the specificity of the antibody was analyzed by searching the cross reactivity with various ANP-related peptides by RIA.
  • the monoclonal antibody obtained was determined to belong to IgG 1 subclass by Ouchterlony's method. Affinity constant, measured by Scatchard method, showed a high affinity, with Ka value against ⁇ -hANP being 6.6 ⁇ 10 10 M -1 (See FIG. 1).
  • the RIA with the use of monoclonal antibody was performed in accordance with the method described in Biochem. Biophys. Res. Commun., 124, 815-821(1984), which involves the use of polyclonal antiserum.
  • reagents employed were always dissolved in 0.1 M phosphate buffer solution (pH 7.0) containing 0.5% gelatin (Merck), 1 mM Na 2 EDTA, 0.2 mM cystine, 0.1% Triton X-100, and 0.01% merthiolate.
  • the specific activity of [ 125 I] ⁇ -hANP was 400-800 ⁇ Ci/ ⁇ g.
  • the binding rate with a tracer was about 30%.
  • the above-mentioned [ 125 I- ⁇ -hANP was prepared by the chloramine T method. That is to say, ⁇ -hANP (1 ⁇ g) was mixed with Na 125 I (1 mCi), to which was added 10 ⁇ l of chloramine T (5.25 mg/ml). Ten seconds after that, 20 ⁇ l of sodium pyrosulfite (4.5 mg/ml) was added. To the mixture was further added 1 ml of 2% gelatin and then the resulting 125 I- ⁇ -hANP was purified with Sep-Pak C18 (Waters Co., Ltd.).
  • the standard curve of ⁇ -hANP determined in RIA with the use of the monoclonal antibody of the present invention, and the cross reactivity with related peptides are shown in FIG. 2.
  • the RIA showed the cross reactivity with ⁇ -rANP at equimolar level, while it showed only a weak reactivity with ⁇ -rANP[3-28] and ⁇ -ANP[1-11], and practically no cross reactivity with ⁇ -ANP[1-6], ⁇ -ANP[4-28], ⁇ -ANP[5-28], and ⁇ -ANP[17-28].
  • the RIA showed 50% and 20% cross reactivity with human atrium-derived ⁇ -hANP and synthetic ⁇ -hANP, respectively (See Table 1).
  • the detection limit of ⁇ -hANP in the standard curve of a ⁇ -hANP shown in FIG. 2 was 0.8 fmol (2.5 pg)/tube, and IC 50 was 8 fmol (25 pg)/tube.
  • the intraassay and interassay variations in this RIA were 10% or less.
  • the antibody is suitable for the measurement not only of human ANP but also of ANP of various experimental animals such as rats and mice. Since KY-ANP-II recognizes the N-terminal of ⁇ -ANP, the antibody makes it possible to specifically measure ⁇ -ANP in the circulating blood.
  • the establishment of the method of measuring ⁇ -hANP by the use of the monoclonal antibody of the present invention has enabled us to easily and accurately diagnose various diseases which are accompanied by abnormality in the balance of body fluid, such as heart diseases, kidney diseases, hypertension (essential and secondary), edematous diseases (cirrhosis, nephrosis, cataplectic edema, etc.), and dehydration, and to follow up the results of treatments.

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US5312744A (en) * 1992-04-30 1994-05-17 Olympus Optical Co., Ltd. Method of immobilizing and preserving an immunologically reactive substance
US5789183A (en) * 1992-08-14 1998-08-04 University Of Arkansas Serological detection and identification of rice blast
CN102768281A (zh) * 2011-05-03 2012-11-07 孙孝芳 用以评估肿瘤增生、侵犯或转移风险的生物标记及方法

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ATE99360T1 (de) 1994-01-15
DE68911713D1 (de) 1994-02-10
JP2724315B2 (ja) 1998-03-09
CA1334076C (en) 1995-01-24
EP0331439B1 (en) 1993-12-29
EP0331439A3 (en) 1991-01-16
EP0331439A2 (en) 1989-09-06
KR890012669A (ko) 1989-09-18
DE68911713T2 (de) 1994-07-14
KR960008672B1 (ko) 1996-06-28
JPH01221399A (ja) 1989-09-04

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